The determination of the hemoglobin content of blood has long been recognized as an invaluable aid to the medical practitioner in the diagnosis of many abnormal conditions. For instance, the condition of iron deficiency anemia in mammals, especially in humans, is well known. This condition, which may be caused by chemical poisoning, infection or disease, is generally characterized by a reduction in the amount of hemoglobin in the blood. Normally, the hemoglobin content in the blood of adult males varies between 12 and 16 grams (g) per 100 milliliters (ml) of blood and in adult females varies between 11 and 15 g per 100 ml of blood. Blood which contains much less than 12 g of hemoglobin per 100 ml of blood is then considered indicative of an anemic condition. In view of the importance of diagnosing such condition, it is considered highly desirable to provide a sensitive, rapid and reliable test for blood hemoglobin.
Various methods and devices are described in the literature for determining the hemoglobin content of blood. Usually, hemoglobin is measured as oxyhemoglobin or is first converted into one of several derivatives, such as alkaline hematin, acid hematin, cyanmethemoglobin or carboxyhemoglobin. The concentration of hemoglobin is then determined by comparing the color or absorbance of the unknown sample with a standard color reference. The method of comparison may be performed by visual matching of colors or by instruments which measure light absorbance of the sample at defined wavelengths. Unfortunately, these methods are often time consuming and require bulky equipment which must be consistently cleaned and maintained to produce reliable results.
Still another procedure is described by T. W. Tallqvist in Z. Klin. Med. 40, 137 (1900) and Arch. Gen. Med. 3, 421 (1900). In this method, a sample of undiluted blood is applied to an absorbent paper and the color of the blood saturated paper is then visually compared with a series of lithographed color standards to estimate the hemoglobin content. Although this procedure is reported to have a margin of error of between 20 and 50%, as discussed by M. M. Wintrobe, Clinical Hematology, Lea and Febiger, Philadelphia, 1961, Page 393, it is still followed by some physicians because of its simplicity. Attempts to quantify the Tallqvist procedure instrumentally by applying light absorption techniques or more recently light reflectance measurements have not been successful due to relatively large margins of error encountered in light absorbance or reflectance measurements.
Several commercially available analytical devices specifically intended for the detection of blood constituents or of bacteria have been tried unsuccessfully by those skilled in the art to estimate hemoglobin content of blood. For example, multilayer devices described in U.S. Pat. Nos. 3,552,925; 3,552,928; or German Offenlegunschrift No. 2,332,760 provide various means, such as porous membranes or filtering layers, for separating red blood corpuscles from blood samples prior to the detection of constituents in the serum; and bacterial detection devices described in U.S. Pat. No. 3,764,480, provide means for detecting surface bacteria by reflectance measurements. It was thought that the hemoglobin content of blood could be estimated be use of these devices and instrumental means. The attempts, however, to quantify these various analytical devices for the detection of hemoglobin content by applying light reflectance or absorption techniques have not been successful due to relatively large margins of error encountered in light reflectance or absorbance measurements.